BR102018008167A2 - process for the preparation of probucol calcogen analogs and their application as a neuroprotective strategy in neurodegenerative processes - Google Patents

Abstract

Process for the preparation of probucol calcogen-analogs and their application as a neuroprotective strategy in neurodegenerative processes The present invention reports the development of organic selenium and sulfur compounds (organocalcogens) analogous to the probucol compound and their use as a neuroprotective strategy in neurodegenerative processes such as such as parkinson's disease, alzheimer's, huntington's, strokes and other neurodegenerative events. specifically reports the synthesis of rc490, rc513, rc363 and rc574 compounds and their use as inhibitors of glutamate and 3-nitropropionic acid-induced neuronal death in ht22 (mouse-derived) immortalized hippocampal neuron cell cultures and the sh-sy5y strain (human neuroblastoma).

Description

PROCESS FOR THE PREPARATION OF PROBUCOL CALCOGEN-ANALOGS AND ITS APPLICATION AS A NEUROPROTECTIVE STRATEGY IN NEURODEGENERATIVE PROCESSES [001] The present invention is comprised in the fields of organic chemistry and pharmacology and consists of the preparation of organocalcogen compounds analogous to the use of probucol, and as a neuroprotective strategy in neurodegenerative processes.

State of the Art [002] Probucol, as well as its succinobucol derivative, has antioxidant and anti-inflammatory properties, in addition to having neuroprotective properties in experimental models of neurodegenerative diseases. In addition, they have important activities in biological systems, among which stand out anti-atherosclerotic and anti-diabetic activities. It should be noted that both compounds (probucol and succinobucol) have already been widely used by humans, and probucol is already used commercially in some countries for the treatment of cardiovascular diseases and dyslipidemias, while succinobucol is being evaluated in clinical trials for the possible treatment of cardiovascular diseases [003] Studies indicate that, in animals, probucol increases neurogenesis, modulates the activity of antioxidant enzymes and has anti-inflammatory properties. In this context, recent studies that demonstrate the neuroprotective effects of probucol in experimental models of Alzheimer's disease and Huntington's disease in

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2/20 rodents. The article “Probucol, a lipid-lowering drug, prevents cognitive and hippocampal synaptic impairments induced by amyloid β peptide in mice”, published in 2012 by Santos, DB; Peres, KC; Ribeiro, RP; Colle, D .; Santos, AA; Moreira, EL; Souza, DO; Figueiredo, CP; Farina, M., shows that probucol was effective to prevent synaptic loss and cognitive impairment induced by amyloid beta-peptide ₁ 4 ₀ (Ae _1-40, which is related to the pathogenesis of Alzheimer's disease); such effects have been attributed, at least in part, to its antioxidant activity.

[004] The article “Probucol Increases Striatal Glutathione Peroxidase Activity and Protects against 3-Nitropropionic Acid-Induced Pro-Oxidative Damage in Rats”, published in 2013 by Colle, D .; Santos, D. B .; Moreira, E.

L.; Hartwig, J. M .; dos Santos, A. A .; Zimmermann, L. T .; Hort, M. A .; Farina,

M., shows that the motor deficit observed in animals submitted to an experimental model of Huntington's disease was prevented by treatment with probucol and this phenomenon was due to the reduction of secondary harmful effects derived from the mitochondrial deficit in the striatal region of the animals.

[005] In addition to probucol, its succinobucol analog also showed neuroprotective properties in experimental models of neurodegenerative disorders. Recent studies have shown that the succinobucol compound showed excellent neuroprotective properties in experimental models of Huntington's disease and Parkinson's disease, as shown in the articles “Succinobucol, a Lipid-Lowering Drug, Protects Against 3Nitropropionic Acid-Induced Mitochondrial Dysfunction and Oxidative Stress in SH -SY5Y Cells via Upregulation of Glutathione Levels and Glutamate Cysteine

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Ligase Activity ”, published in 2016 by Colle, D., Santos, DB, Hartwig, JM, Godoi, M, Engel, DF, de Bem, AF, Braga, AL, Farina, M.,“ Succinobucol, a NonStatin Hypocholesterolemic Drug , Prevents Premotor Symptoms and Nigrostriatal Neurodegeneration in an Experimental Model of Parkinson's Disease ”, published in 2016.

[006] Recently, some studies have reported the importance of the phenolic portion derived from probucol as a group responsible for some of its biological actions. These studies also point to the possibility of structural alteration of this compound in order to obtain better pharmacological effects and / or less adverse effects. In this context, Ladopoulou et al. showed, in the publication "New Multifunctional Di-tertbutylphenoloctahydro (pyrido / benz) oxazine Derivatives with Antioxidant, Antihyperlipidemic, and Antidiabetic Action" - Med. Chem. 2013, 56, 3330, that a probucol analog compound, with modifications in the side chain, has better antioxidant, anti-hyperglycemic and antidiabetic activities when compared to other compounds in this class. Other studies have shown that simplified probucol analogues, such as disulfides or selenesulfides, maintain their action in the treatment of atherosclerosis, suggesting the need for the phenolic portion and the sulfur atom as important structural patterns for some biological activities.

[007] The patent document WO9940911 - "Use of selenium compounds for preventing and treating alzheimer disease" deposited on 08/19/1999, describes a series of selenides that are indicated in their acceptable pharmaceutical forms in the treatment of Alzheimer's disease.

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4/20 [008] Patent document EP1774972 - “Use of selenium yeasts in the treatment of Alzheimers disease”, published on 04/18/2007, demonstrates the use of selenium-rich compositions (Sel-Plex) for prophylaxis and treatment of neurodegenerative disease, as it is able to alter the expression of genes involved in the disease, leading to an improvement in the patient.

[009] The current state of the art indicates that changes in the structure of probucol may lead to new compounds with better bioavailability, better pharmacological effects and fewer adverse effects.

[0010] The present invention reports the development of organic compounds of selenium and sulfur (organocalcogens) analogous to the compound probucol and its use as a neuroprotective strategy in neurodegenerative processes, such as Parkinson's disease, Alzheimer's, Huntington's, strokes and other neurodegenerative events .

[0011] Specifically, it reports the synthesis of compounds RC490, RC513, RC363 and RC574, and their use as inhibitors of neuronal death induced by glutamate and 3-nitropopionic acid in cultures of neuronal cells of the strain HT22 (immortalized hippocampal neuron, derived mouse) and SH-SY5Y (human neuroblastoma). Results are also presented that allow to evaluate possible mechanisms related to the observed neuroprotective effects.

[0012] The probucol analogues, RC490, RC513, RC363 and RC574, were designed from the structure of probucol by molecular simplification, stiffening of the side chain and bioisosterism between the sulfur and selenium atoms.

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Probucol Structure

Detailed Description of the Invention [0013] The 2,6-di-tert-butyl-4-selenocyanatophenol selenocyanate, called RC490, can be prepared from the reaction between 2,6-di-tert-butyl-phenol and dicycate triselenyl (NCSeSeSeCN) as an electrophilic selenium species, according to Reaction 1. Triselenyl dicycate should be prepared preferably in situ, just before use, preferably using dimethyl sulfoxide as the reaction solvent, using room temperature to conduct the reaction. The preparation of triselenyl dicyanate is carried out by reacting malononitrile and SeO ₂ , preferably in the proportion of 1: 2 equivalents.

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NCSeSeSeCN

DMSO t.a

0.5h 76%

Reaction 1 [0014] The RC490 can be synthesized using the procedure described below. In a flask mix malononitrile (1 equivalent), DMSO (1.2 mL / mmol) and SeO ₂ (2 equivalents), stir the mixture for 20 minutes at room temperature and then add 2,6-di-tert-butylphenol (1 equivalent). After 30 minutes, dilute the mixture with AcOEt and extract with water (5x2 mL / mmol) and brine (1x1 mL / mmol), dry the organic phase over MgSO ₄ , filter and evaporate. Purify the reaction crude by column chromatography using hexane as the eluent.

[0015] The synthesis of the RC490 can be performed, preferably, through the one described below. In a flask mix malononitrile (5 mmol), DMSO (6 mL) and SeO ₂ (10 mmol). Stir the mixture for 20 minutes at room temperature and then add 2,6-di-tert-butylphenol (5 mmol). After 30 minutes, dilute the mixture with AcOEt and extract with water (5x10 mL) and brine (1x 5 mL). Dry the organic phase over MgSO ₄ , filter and evaporate. Purify the reaction crude by column chromatography using hexane as the eluent generating 3.82 mmol (76% yield) of the product.

[0016] The 4,4'-diselanediilbis diselenide (2,6-di-tert-butylphenol), called RC513, can be prepared from the RC490 selenocyanate by deprotecting the selenium atom, Reaction 2. Deprotection can be conducted in an acidic environment, using H ₃ PO ₄ , or can be conducted in an

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7/20 basic, using for example K2CO3 or KOH, or it can also be conducted in a reducing medium, using for example NaBH ₄ or LiAIH ₄ . Deprotection is preferably carried out using NaBH ₄ in an ethereal solvent. The reaction is preferably carried out at room temperature. The reaction must be conducted in the presence of atmospheric (open) air, as oxidation occurs in situ to form the Se-Se bond.

NaBH,

THF. t to 10niin, 93%

Reaction 2 [0017] The synthesis of the RC513 can be performed using the procedure described below. Solubilize 1 equivalent of RC490, 2,6-di-tert-butyl-4selenocyanatophenol, in THF (9 mL / mmol) and add 1 equivalent of NaBH ₄ . Stir the mixture at room temperature for 10 minutes and add 9 mL / mmol of saturated NH ₄ CI solution; After 10 minutes, dilute the reaction medium with AcOEt and extract with water (3x15 mL / mmol) and brine (1x9 mL / mmol); Dry the organic phase over MgSO ₄ , filter and evaporate, generating the compound without the need for further purification;

[0018] The synthesis of the RC513 can be performed, preferably, through the procedure described below. Solubilize RC490, 2,6-di-tert-butyl4-selenocyanatophenol (0.32 mmol), in THF 3 mL and then add NaBH ₄ (0.32 mmol). Stir the mixture at room temperature for 10 minutes and then add 3 mL of saturated NH ₄ CI solution. After 10 minutes, dilute the medium

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8/20 reaction with AcOEt and extract with water (3x5 mL) and brine (1x3 mL). Dry the organic phase over MgSO4, filter and evaporate. Thus, 0.15 mmol (93% yield) of the compound is obtained without the need for further purification.

[0019] The 2,6-di-tert-butyl-4- (thiophen-ylselanyl) phenol selenide, called RC574, can be obtained from the RC490 selenocyanate, according to Reaction 3. The thiophene is treated with butyl lithium, BuLi , preferably in an ethereal solvent, for deprotonation of the thiophene and formation of the lithium derivative of the thiophene. This process is preferably carried out at low temperatures, preferably -78 ° C using tetrahydrofuran as the solvent. After deprotonation the lithium derivative of the thiophene is reacted with the RC490 selenocyanate as an electrophilic selenium species to form the target selenide. Preferably the lithium derivative of thiophene is prepared in situ, used immediately after its preparation and kept at low temperatures under an inert atmosphere.

Reaction 3 [0020] The synthesis of RC574 can be performed, using the procedure described below. In a flask, under an inert atmosphere, add 2.5 mL / mmol of THF and 1 equivalent of thiophene, and cool the reaction mixture to -78 ° C; Add a BuLi solution (1.8M, 1 equivalent) dropwise and stir for 1h; Add the RC490 compound and let the system cool with stirring for 30 min; Extract the reaction mixture with AcOEt, water and brine, and reaction crude

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9/20 can be purified by column chromatography using hexane as the eluent.

[0021] The synthesis of RC 574 can be performed, preferably, through the procedure described below. In a flask, under an inert atmosphere, add THF (5 mL) and thiophene (2 mmol) and cool the reaction mixture to -78 ° C. Add BuLi solution (1.8M, 2 mmol) over this mixture and stir for 1h. Still at -78 ° C add compound RC490 and then let the system cool under stirring for 30 min. At the end, extract the reaction mixture with AcOEt, water and brine. The reaction crude is sufficiently pure, but can be purified by column chromatography using hexane as the eluant.

[0022] The 2,6-di-tert-butyl-4- (thiophen-ylthio) phenol sulfide, called RC363, can be obtained from the thiophene, according to Reaction 4. The thiophene is treated with butyl lithium, BuLi, preferably in an ethereal solvent, for deprotonation of the thiophene and formation of the lithium derivative of the thiophene. This process is preferably carried out at low temperatures, preferably at -78 ° C using tetrahydrofuran as the solvent. After deprotonation, the lithium derivative of the thiophene is reacted with elemental sulfur to form in situ the thiolate derived from the thiophene. Preferably the lithium derivative of thiophene is prepared in situ, used immediately after its preparation and kept at low temperatures under an inert atmosphere. The thiophene derived from the thiophene is then reacted with 4-bromo-2,6-di-tert-butyl-phenol in the presence of a copper salt, preferably copper iodide, Cul. Preferably this reaction is carried out under high temperatures, preferably under reflux of tetrahydrofuran and in an inert atmosphere. Thiophene-derived thiolate can be

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10/20 isolated in the form of thiol, but preferably it is prepared just before its use and is used in situ in the subsequent reaction.

1. BuLi, THF,

O - ⁷⁸ ° ^C ' ^1h r

2. S °, THF,

-78 ° C-RT, 1h

Reaction 4 [0023] The synthesis of the RC363 can be performed, using the procedure described below. In a flask, under an inert atmosphere, add 1 equivalent of thiophene and 1.5 mL / mmol of dry THF, and cool the solution to -78 ° C; Add a BuLi solution (1.8 M, 1 equivalent) dropwise; After the addition, stir the mixture at -78 ° C for 1h; Add 1 equivalent of S °; Allow the suspension to come to room temperature and stir until the elemental chalcogen is consumed; Add bromide (1 equivalent), CuI (0.1 equivalent) and place the reaction mixture under reflux until the consumption of the bromide of the reaction mixture by the CCD is indicated; Cool the reaction medium, extracted with AcOEt, water, brine, dried with MgSO ₄ , filter and evaporate; Purify the reaction crude by column chromatography using hexane as the eluent;

[0024] The synthesis of the RC363 can be performed, preferably, through the procedure described below. In a flask, under an inert atmosphere, add thiophene (4 mmol) and dry THF (6 mL). Cool this solution to -78 ^o C and add a BuLi solution (1.8 M, 4 mmol) dropwise. After the addition, stir the mixture at -78 ^o C for 1 h and then add S ^o (4 mmol). Leave the

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11/20 suspension come at room temperature and stir until the consumption of elemental chalcogen. Add bromide (4 mmol), CuI (0.4 mmol) and place the reaction mixture under reflux until the consumption of the bromide of the reaction mixture by the CCD is indicated. Cool the reaction medium, extracted with AcOEt, water, brine, dried with MgSO ₄ , filter and evaporate. Purify the reaction crude by column chromatography using hexane as the eluent.

[0025] The compounds described in the present invention have potential as therapeutic strategies in neurodegenerative processes, as they are neuroprotective against the death of neuronal cells (immortalized murine neurons and human neuroblastoma) exposed to harmful molecules that lead to mitochondrial dysfunction, which represents a phenomenon common to many neurodegenerative disorders, such as Parkinson's, Alzheimer's and Huntington's. The protective effects observed depend on the chemical structure of the compounds, indicating specific effects for the different harmful conditions.

[0026] The neuroprotective effects and the specificity of the compounds can be observed in the tests described below.

[0027] Neuronal cells of the HT22 lineage were exposed to high concentrations of glutamate, which represents a model widely used for the induction of neurodegeneration and screening of molecules with neuroprotective properties. Figures 1 and 2 show the viability of HT22 neuronal cells exposed to high concentrations of glutamate, which was assessed based on the metabolic capacity of the cells to metabolize the compound MTT (3- (4,5-dimethylthiazole-2yl) -2,5-diphenyl bromide

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12/20 tetrazoline) and the rupture of the cell membrane and the consequent incorporation of the propidium iodide compound into the cellular DNA. It is observed that glutamate significantly reduced cell viability and that compounds RC363 and RC574 were able to protect against the deleterious effects induced by glutamate, Fig. 1.

[0028] The results expressed in Fig. 1, show the effect of treatment with probucol (PB) and its unpublished derivatives on the viability of HT22 cells exposed to glutamate. The cells were treated with the compounds at a concentration of 3 μΜ for 24 hours in DMEM medium with 10% (v / v) fetal bovine serum (SFB). Subsequently, the medium was exchanged for DMEM with 3% (v / v) SFB and the cells were exposed to glutamate at a concentration of 5 mM or 10 mM. Cell viability was assessed 24 h after treatment with glutamate by reducing MTT. Results expressed as a percentage of the control (untreated cells, 100%). Each bar represents the mean ± standard error of the mean (SEM) of 5 independent experiments. ** p <0.01, *** p <0.001, **** p <0.0001 indicate the difference when compared to the group treated with their respective control (treated only with vehicle or compound). + p <0.05, ++ p <0.01, +++ p <0.001 indicate difference when compared to the group treated only with glutamate (two-way ANOVA followed by the Tukey post hoc test).

[0029] It is observed that glutamate significantly increased the incorporation of propidium iodide (indicative of cell death) and that compounds RC363 and RC574 were able to protect against the effects of glutamate, Fig. 2. Both compounds, RC363 and RC574, showed effects

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13/20 superior neuroprotective when compared to the Probucol (PB) compound, which has already shown significant neuroprotective properties as previously mentioned.

[0030] The results expressed in Fig. 2, show the effect of treatment with probucol (PB) and its unpublished derivatives on the death of HT22 cells exposed to glutamate. The cells were treated with the compounds at a concentration of 3 μΜ for 24 hours in DMEM medium with 10% (v / v) fetal bovine serum (SFB). Subsequently, the medium was exchanged for DMEM with 3% (v / v) SFB and the cells were exposed to glutamate at a concentration of 5 mM or 10 mM. Cell death was assessed 24 h after treatment with glutamate through the incorporation of PI. Results expressed as percentage of cells treated with 0.2% Triton (100% death). Each bar represents the mean ± standard error of the mean (SEM) of 5 independent experiments. * p <0.05, **** p <0.0001 indicate the difference when compared to the group treated with their respective control (treated only with vehicle or compound). ++ p <0.01, ++++ p <0.0001 indicate difference when compared to the group treated with glutamate only (two-way ANOVA followed by the Tukey post hoc test).

[0031] The same cell viability tests were performed on HT22 cells, however, evaluating the possible neuroprotective effects of compounds RC490 and RC513, Fig. 3 and 4. A lower concentration (500 nM) of the compounds was used when compared to that used in the experiments related to Figures 1 and 2 (3 mM). It is observed that glutamate significantly reduced cell viability and that compounds RC490 and

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RC513 were able to protect against the deleterious effects induced by glutamate, Fig. 3. Also, it is observed that glutamate significantly increased the incorporation of propidium iodide (indicative of cell death) and that compounds RC490 and RC513 were able to protect against the effects of glutamate, Fig. 4. Both compounds, RC490 and RC513, have superior neuroprotective effects when compared to the standard compound Probucol (PB).

[0032] The results expressed in Fig. 3 show the effect of treatment with probucol (PB) and its derivatives on the viability of HT22 cells exposed to glutamate. The cells were treated with the compounds at a concentration of 0.5 μΜ for 24 hours in DMEM medium with 10% (v / v) fetal bovine serum (SFB). Subsequently, the medium was exchanged for DMEM with 3% (v / v) SFB and the cells were exposed to glutamate at a concentration of 3 mM. Cell viability was assessed 24 h after treatment with glutamate by reducing MTT. Results expressed as a percentage of the control (untreated cells, 100%). Each bar represents the mean ± standard error of the mean (SEM) of 7 independent experiments. ** p <0.01, *** p <0.001, **** p <0.0001 indicate the difference when compared to the group treated with their respective control (treated only with vehicle or compound). + p <.05, ++ p <0.01, +++ p <0.001 indicate difference when compared to the group treated with glutamate only (two-way ANOVA followed by the Tukey post hoc test).

[0033] The results expressed in Fig. 4 show the effect of treatment with probucol (PB) and its derivatives on the death of exposed HT22 cells

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15/20 to glutamate. The cells were treated with the compounds at a concentration of 0.5 pM for 24 hours in DMEM medium with 10% (v / v) fetal bovine serum (SFB). Subsequently, the medium was exchanged for DMEM with 3% (v / v) SFB and the cells were exposed to glutamate at a concentration of 3 mM. Cell death was assessed 24 h after treatment with glutamate through the incorporation of PI. Results expressed as percentage of cells treated with 0.2% Triton (100% fluorescence). Each bar represents the mean ± standard error of the mean (SEM) of 7 independent experiments. * p <0.05, **** p <0.0001 indicate the difference when compared to the group treated with their respective control (treated only with vehicle or compound). ++ p <0.01, ++++ p <0.0001 indicate difference when compared to the group treated with glutamate only (two-way ANOVA followed by the Tukey post hoc test).

[0034] In order to understand the mechanisms related to the protective effects of compounds analogous to probucol, the possible ability of the compounds to prevent the reduction of glutathione levels was evaluated (evaluated by the technique that measures non-protein thiols, from English nonprotein thiols, NPSH) and the generation of reactive species resulting from exposure to glutamate. It is observed that glutamate was able to decrease the concentration of the antioxidant molecule glutathione in 6 hours after treatment, Fig. 5 and 6. However, the compounds derived from probucol, which protected against decreased viability in 24h, Fig. 1 to 4, were not able to prevent a decrease in glutathione levels, Fig. 5 and 6.

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16/20 [0035] The results expressed in Fig. 5 show the effect of treatment with probucol (PB) and its derivatives on the NPSH levels of HT22 cells exposed to glutamate. The cells were treated with the compounds at a concentration of 3 μΜ for 24 hours in DMEM medium with 10% (v / v) fetal bovine serum (SFB). Subsequently, the medium was exchanged for DMEM with 3% (v / v) SFB and the cells were exposed to glutamate at a concentration of 5 mM. NPSH levels were assessed 6 h after treatment with glutamate. Results expressed as a percentage of the control (untreated cells, 100%). Each bar represents the mean ± standard error of the mean (SEM) of 5 independent experiments. * p <0.05, *** p <0.001, **** p <0.0001 indicate the difference when compared to the group treated with their respective control (treated only with vehicle or compound) (two-way ANOVA followed by the test post hoc Tukey).

[0036] The results expressed in Fig. 6 show the effect of treatment with probucol (PB) and its derivatives on the NPSH levels of HT22 cells exposed to glutamate. The cells were treated with the compounds at a concentration of 0.5 μM for 24 hours in DMEM medium with 10% (v / v) fetal bovine serum (SFB). Subsequently, the medium was exchanged for DMEM with 3% (v / v) SFB and the cells were exposed to glutamate at a concentration of 3 mM. NPSH levels were assessed 6 h after treatment with glutamate. Results expressed as a percentage of the control (untreated cells, 100%). Each bar represents the mean ± standard error of the mean (SEM) of 5 independent experiments. * p <0.05, *** p <0.001, **** p <0.0001 indicate the difference when compared with the group treated with their respective

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17/20 control (treated with vehicle or compound only). (Two-way ANOVA followed by the Tukey post hoc test).

[0037] Some of the compounds similar to probucol were able to prevent the generation of reactive species resulting from exposure to glutamate, indicating potent antioxidant effects that do not depend on the endogenous molecule glutathione, Figs 7 and 8.

[0038] The results expressed in Fig. 7 show the effect of treatment with probucol (PB) and its derivatives on the levels of Reactive Oxygen Species (ROS) in HT22 cells exposed to glutamate. The cells were treated with the compounds at a concentration of 3 μΜ for 24 hours in DMEM medium with 10% (v / v) fetal bovine serum (SFB). Subsequently, the medium was exchanged for DMEM with 3% (v / v) SFB and the cells were exposed to glutamate at a concentration of 5 mM. ROS levels were assessed 6 h after treatment with glutamate by detecting DCF fluorescence. Results expressed as a percentage of the control (untreated cells, 100%). Each bar represents the mean ± standard error of the mean (SEM) of 5 independent experiments. * p <0.05, ** p <0.01, **** p <0.0001 indicate the difference when compared to the group treated with their respective control (treated only with vehicle or compound). ++ p <0.01, ++++ p <0.0001 indicate difference when compared to the group treated with glutamate only (two-way ANOVA followed by the Tukey post hoc test).

[0039] The results expressed in Fig. 8 show the effect of treatment with probucol (PB) and its derivatives on the levels of Reactive Oxygen Species (ROS) in HT22 cells exposed to glutamate. The cells were

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18/20 treated with the compounds at a concentration of 0.5 μΜ for 24 hours in DMEM medium with 10% (v / v) fetal bovine serum (SFB). Subsequently, the medium was exchanged for DMEM with 3% (v / v) SFB and the cells were exposed to glutamate at a concentration of 3 mM. ROS levels were assessed 6 h after treatment with glutamate by detecting DCF fluorescence. Results expressed as a percentage of the control (untreated cells, 100%). Each bar represents the mean ± standard error of the mean (SEM) of 7 independent experiments. * p <0.05, ** p <0.01, **** p <0.0001 indicate the difference when compared to the group treated with their respective control (treated only with vehicle or compound). ++ p <0.01, ++++ p <0.0001 indicate difference when compared to the group treated with glutamate only (two-way ANOVA followed by the Tukey post hoc test).

[0040] In addition to the experiments with immortalized neuronal cells derived from the mouse hippocampus (HT22), the possible neuroprotective effects of the novel compounds analogous to probucol were investigated in neuronal cells of the SH-SY5Y (human neuroblastoma) lineage, however, exposed to the compound 3-nitropropionic acid (3-NP), which has been used in experimental models of Huntington's disease.

[0041] Neuronal cells of the SH-SY5Y lineage were treated with compounds derived from probucol and, subsequently, were exposed to compound 3-NP. Figures 9 and 10 show the viability of SH-SY5Y neuronal cells exposed to the 3-NP compound, which was evaluated based on the metabolic capacity of the cells to metabolize the MTT compound, as previously described. It is observed that the compound 3-NP reduced

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19/20 significantly reduced cell viability and that compounds RC363 and RC574 were not able to protect against the deleterious effects induced by 3NP, Fig. 9.

[0042] The results expressed in Fig. 9 show the effect of treatment with probucol (PB) and its derivatives on the viability of SH-SY5Y cells exposed to 3-nitropropionic acid (3-NP). The cells were treated with the compounds at a concentration of 2-3 μΜ for 6 days in DMEM medium with 10% (v / v) fetal bovine serum (SFB). Subsequently, the cells were exposed to 3-NP at a concentration of 3 mM. Cell viability was assessed 24 h after treatment with 3-NP by reducing MTT. Results expressed as a percentage of the control (untreated cells, 100%). Each bar represents the mean ± standard error of the mean (SEM) of 5 independent experiments. * p <0.05, *** p <0.001 indicate the difference when compared to the group treated with their respective control (treated only with vehicle or compound) (two-way ANOVA followed by the Tukey post hoc test).

[0043] The compound 3-NP significantly reduced cell viability and compounds RC490 and RC513 are able to protect against the deleterious effects induced by 3-NP. Both compounds, RC490 and RC513, have superior neuroprotective effects when compared to the standard compound probucol, PB.

[0044] The results expressed in Fig. 10 show the effect of treatment with probucol (PB) and its derivatives on the viability of SH-SY5Y cells exposed to 3-nitropropionic acid (3-NP). The cells were treated with the compounds at a concentration of 0.5 μM for 6 days in DMEM medium

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20/20 with 10% (v / v) fetal bovine serum (SFB). Subsequently, the cells were exposed to 3-NP at a concentration of 3 mM. Cell viability was assessed 24 h after treatment with 3-NP by reducing MTT. Results expressed as a percentage of the control (untreated cells, 100%). Each bar represents the mean ± standard error of the mean (SEM) of 5 independent experiments. * p <0.05, *** p <0.001 indicate the difference when compared to the group treated with their respective control (treated only with vehicle or compound) (two-way ANOVA followed by the Tukey post hoc test).

[0045] The aforementioned results demonstrate that probucol calcogeno-analogs, described in the present invention, can be considered potential pharmacological / neuroprotective strategies in neurodegenerative processes, example: Parkinson's disease, Alzheimer's, Huntington's, strokes and other neurodegenerative events . The compounds used protected neuronal cells from cell death induced by two compounds that act, under the mentioned conditions, as neurotoxins. It should be noted that the two compounds used as neurotoxins (glutamate and 3-nitropropionic acid) cause deleterious effects in neuronal cells by activating mechanisms of oxidative stress and mitochondrial damage, which represent common to many neurodegenerative disorders, such as Parkinson, Alzheimer and Huntington . As previously mentioned, the protective effects observed depended on the chemical structure of the compounds, indicating specific effects for the different harmful conditions.

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Claims (21)

1. PROBUCOL CALCOGEN-ANALOGS characterized by having a base composition demonstrated in the formula: forming organic selenium compounds (organocalcogens). 2. PROBUCOL CALCOGEN-ANALOG, according to claim 1, characterized in that R represents CN cyanide, and the molecule has a 2,6-di-tert-butyl-4-selenocyanatophenol composition, called RC490; 3. PROBUCOL CALCOGEN-ANALOG according to claim 1, characterized in that R represents the same base selenide molecule: and the molecule has a composition 4,4'-diselanediilbis (2,6-di-tert-butylphenol), called RC513; 4. PROBUCOL CALCOGEN-ANALOG, according to claim 1, characterized in that R represents the thiophene, C4H4S, and the molecule has the composition 2,6-di-tert-butyl-4- (thiophen-ylselanyl) phenol, called RC574; Petition 870180032819, of 04/23/2018, p. 32/94 2/10 5. PROBUCOL CALCOGEN-ANALOG characterized by having 2,6-di-tert-butyl ~ 4- (thiophen-ylthio) phenol composition, called RC363, demonstrated in the formula: 6. PROCEDURE FOR THE PREPARATION OF PROBUCOL CALCOGENOANALOGUES, according to claim 2, characterized by the RC490 synthesis being based on the reaction between 2,6-di-tertbutyl-phenol and triselenyl dicycate (NCSeSeSeCN) as an electrophilic species selenium, and the triselenyl dicyanate is preferably prepared in situ, just before its use, preferably using dimethyl sulfoxide as the reaction solvent, using room temperature to conduct the reaction, and the preparation of the triselenyl dicianate is carried out by reacting malononitrile and SeO2, preferably in the proportion of 1: 2 equivalents. 7. PROCESS FOR THE PREPARATION OF PROBUCOL CALCOGENOANALOGUE, according to claim 2, characterized by the RC490 being obtained, preferably, through the procedure described below: • In a flask mix malononitrile (1 equivalent), DMSO (1.2 mL / mmol) and SeO2 (2 equivalents), • Shake the mixture for 20 minutes at room temperature and then, Petition 870180032819, of 04/23/2018, p. 33/94 3/10 • add 2,6-di-tert-butylphenol (1 equivalent), • After 30 minutes, dilute the mixture with AcOEt and extract with water (5 x 2 mL / mmol) and brine (1x 1 mL / mmol) , • Dry the organic phase over MgSO4, filter and evaporate, • Purify the reaction crude by column chromatography using hexane as the eluent. 8. PROCESS FOR THE PREPARATION OF PROBUCOL CALCOGENOANALOGUE, according to claim 2, characterized by the RC490 being obtained, preferably, through the procedure described below: • In a flask mix malononitrile (5 mmol), DMSO (6 mL) and SeO2 (10 mmol), • Stir the mixture for 20 minutes at room temperature and then, • add 2,6-di-tert-butylphenol (5 mmol ), • After 30 minutes, dilute the mixture with AcOEt and extract with water (5 x 10 mL) and brine (1x 5 mL), • Dry the organic phase over MgSO4, filter and evaporate, • Purify the reaction crude by chromatography on column using hexane as eluent generating 3.82 mmol of the product. 9. PROCESS FOR THE PREPARATION OF CALCOGENO- PROBUCOL ANALOGS, according to claim 3, characterized by the composition 4,4'-diselanediilbis (2,6-di-tert-butylphenol), called RC513, be obtained by the deprotection of the RC490 selenocyanate, demonstrated in claim 2, through the selenium atom, and the deprotection is conducted Petition 870180032819, of 04/23/2018, p. 34/94 4/10 in an acidic medium, using H3PO4, or be conducted in a basic medium, using for example K2CO3 or KOH, or still be conducted in a reducing medium, using for example NaBH4 or LiAlH4, preferably deprotection is conducted using NaBH4 in an ethereal solvent , and the reaction is conducted preferably at room temperature and in the presence of atmospheric (open) air, with oxidation in situ and formation of the Se-Se bond. 10. PROCEDURE FOR THE PREPARATION OF PROBUCOL CALCOGENOANALOGUE, according to claim 3, characterized in that RC513 is preferably obtained through the procedure described below: • Solubilize 1 equivalent of RC490, 2,6-di-tert-butyl-4-selenocyanatophenol, in THF (9 mL / mmol); • Add 1 equivalent of NaBH4; • Stir the mixture at room temperature for 10 minutes; • Add 9 mL / mmol of saturated NH4Cl solution; • After 10 minutes, dilute the reaction medium with AcOEt and extract with water (3 x 15 mL / mmol) and brine (1 x 9 mL / mmol); • Dry the organic phase over MgSO4, filter and evaporate, generating the compound without the need for further purification; 11. PROCEDURE FOR THE PREPARATION OF PROBUCOL CALCOGENOANALOGUE, according to claim 3, characterized in that RC513 is preferably obtained through the procedure described below: Petition 870180032819, of 04/23/2018, p. 35/94 5/10 • Solubilize 0.32 mmol of RC490, 2,6-di-tert-butyl-4-selenocyanatophenol in THF 3 mL; • Add 0.32 mmol of NaBH < • Stir the mixture at room temperature for 10 minutes; • Add 3 mL of saturated NH4Cl solution; • After 10 minutes, dilute the reaction medium with AcOEt and extract with water (3 x 5 ml) and brine (1 x 3 ml); • Dry the organic phase over MgSO4, filter and evaporate, generating 0.15 mmol of the compound without the need for further purification; 12. PROCESS FOR THE PREPARATION OF PROBUCOL CALCOGENOANALOGUE, according to claim 4, characterized by the composition 2,6-di-tert-butyl-4- (thiophen-ylselanyl) phenol, called RC574, to be obtained by treating the thiophene with butyl lithium, BuLi, preferably in an ethereal solvent, for deprotonation of the thiophene and formation of the lithium derivative of the thiophene, preferably at low temperatures, preferably 78 ° C using tetrahydrofuran as the solvent, and after deprotonation the lithium derivative of the thiophene is reacted with the selenocyanato RC490 as an electrophilic selenium species to form the target selenide, preferably the lithium derivative of thiophene be prepared in situ, used right after its preparation and kept at low temperatures under an inert atmosphere. 13. PROCEDURE FOR THE PREPARATION OF PROBUCOL CALCOGENOANALOGUE, according to claim 4, characterized in that RC574 is preferably obtained through the procedure described below: Petition 870180032819, of 04/23/2018, p. 36/94 6/10 • In a flask, under an inert atmosphere, add 2.5 mL / mmol of THF and 1 equivalent of thiophene, and cool the reaction mixture to 78 ° C; • Add a BuLi solution (1.8M, 1 equivalent) drop by drop; • Shake for 1h; • Add the RC490 compound and let the system cool with stirring for 30 min; • Extract the reaction mixture with AcOEt, water and brine, and the reaction crude can be purified by column chromatography using hexane as an eluent. 14. PROCEDURE FOR THE PREPARATION OF PROBUCOL CALCOGENOANALOGUE, according to claim 4, characterized in that RC574 is preferably obtained through the procedure described below: • In a flask, under an inert atmosphere, add 5mL of THF and 2 mmol of thiophene, and cool the reaction mixture to -78 ° C; • Add a BuLi solution (1.8M, 2 mmol) dropwise; • Shake for 1h; • Add the RC490 compound and let the system cool with stirring for 30 min; • Extract the reaction mixture with AcOEt, water and brine, and the reaction crude can be purified by column chromatography using hexane as an eluent. Petition 870180032819, of 04/23/2018, p. 37/94 7/10 15. PROCEDURE FOR THE PREPARATION OF PROBUCOL CALCOGENOANALOGUE, according to claim 5, characterized in that 2,6-di-tert-butyl-4- (thiophenylthio) phenol, called RC363, is obtained from thiophene and thiophene be treated with butyl lithium, BuLi, preferably in an ethereal solvent, with deprotonation of the thiophene and formation of the lithium derivative of the thiophene and, preferably this process is carried out at low temperatures, preferably at -78 ° C using tetrahydrofuran as solvent, and after deprotonation the lithium derivative of the thiophene is reacted with elemental sulfur for in situ formation of the thiolate derived from the thiophene, preferably the lithium derivative of the thiophene is prepared in situ, used immediately after its preparation and kept at low temperatures under an inert atmosphere, and the Thiol derivative thiolate is then reacted with 4-bromo-2,6-di-tert-butylphenol in the presence of a copper salt, preferably copper iodide, Cul, and, preferably preferably, this reaction is conducted under high temperatures, preferably under reflux of tetrahydrofuran and in an inert atmosphere, and the thiol derivative of the thiophene is isolated in the form of thiol, but it is preferably prepared just before its use and is used in situ in the subsequent reaction . 16. PROCEDURE FOR THE PREPARATION OF PROBUCOL CALCOGENOANALOGUE, according to claim 5, characterized in that RC363 is preferably obtained through the procedure described below: • In a flask, under an inert atmosphere, add 1 equivalent of thiophene and 1.5 mL / mmol of dry THF, and cool the solution to -78 ° C; Petition 870180032819, of 04/23/2018, p. 38/94 8/10 • Add a BuLi solution (1.8 M, 1 equivalent) dropwise; • After adding, stir the mixture at -78 ° C for 1h; • Add 1 equivalent of S °; • Allow the suspension to come to room temperature and stir until the elemental chalcogen is consumed; • Add bromide (1 equivalent), CuI (0.1 equivalent) and place the reaction mixture under reflux until the consumption of the bromide of the reaction mixture by the CCD is indicated; • Cool the reaction medium, extracted with AcOEt, water, brine, dried with MgSO4, filter and evaporate; • Purify the reaction crude through column chromatography using hexane as an eluent; 17. PROCEDURE FOR THE PREPARATION OF PROBUCOL CALCOGENOANALOGUE, according to claim 5, characterized in that RC363 is preferably obtained through the procedure described below: • In a flask, under an inert atmosphere, add 4 mmol of thiophene and 6 mL of dry THF, and cool the solution to -78 ° C; • Add a BuLi solution (1.8 M, 4 mmol) dropwise; • After adding, stir the mixture at -78 ° C for 1h; • Add 4 mmol of S °; • Allow the suspension to come to room temperature and stir until the elemental chalcogen is consumed; Petition 870180032819, of 04/23/2018, p. 39/94 9/10 • Add bromide (4 mmol), CuI (0.4 mmol) and place the reaction mixture under reflux until the consumption of the bromide of the reaction mixture by the CCD is indicated; • Cool the reaction medium, extracted with AcOEt, water, brine, dried with MgSO4, filter and evaporate; • Purify the reaction crude through column chromatography using hexane as an eluent; 18. APPLICATION AS A NEUROPROTECTIVE STRATEGY IN NEURODEGENERATIVE PROCESSES OF PROBUCOL CALCOGEN-ANALOG, characterized by 2,6-di-tert-butyl-4-selenocyanatophenol, called RC490, acting as a neuroprotective therapeutic agent in neurodegenerative conditions, such as Parkinson's disease, Alzheimer's, Huntington's, stroke and other neurodegenerative events. 19. APPLICATION AS A NEUROPROTECTIVE STRATEGY IN PROBUCOL NEURODEGENERATIVE CALCOGEN-ANALOG PROCESSES, characterized by 4,4'-diselanediilbis (2,6-di-tert-butylphenol), called RC513, to act as a neuroprotective therapeutic agent in neurodegenerative disease Parkinson's, Alzheimer's, Huntington's, strokes and other neurodegenerative events. 20. APPLICATION AS A NEUROPROTECTIVE STRATEGY IN NEURODEGENERATIVE PROCESSES OF PROBUCOL CALCOGEN-ANALOG, characterized by 2,6-di-tert-butyl-4- (thiophen-ylselanil) phenol, called RC574, acting as a neuroprotective therapeutic agent under conditions Petition 870180032819, of 04/23/2018, p. 40/94 10/10 neurodegenerative, such as Parkinson's disease, Alzheimer's, Huntington's, strokes and other neurodegenerative events. 21. APPLICATION AS A NEUROPROTECTIVE STRATEGY IN NEURODEGENERATIVE PROCESSES OF PROBUCOL CALCOGEN-ANALOG, characterized by 2,6-di-tert-butyl-4- (thiophen-ylthium) phenol, called RC363, to act as a neuroprotective therapeutic agent in neurodegenerative conditions, Parkinson's disease, Alzheimer's, Huntington's, strokes and other neurodegenerative events.